Hematopoiesis involves the differentiation and proliferation of stem cells, which are specified during embryogenesis. The vasculature and hematopoietic cells appear closely associated in embryonic sites and are thought to derive from a common cell known as the hemangioblast. Transcription factors such as SCL and LM02 are critical for both vasculogenesis and hematopoiesis. To further our understanding of these molecular programs, we formed a consortium of investigators with expertise in stem cell biology and genomics. Our aim is to create cDNA libraries from stem cell enriched populations from the mouse and zebrafish. The stem cell sources include blast colonies from differentiated murine ES (embryonic stem) cells and marrow cells from transgenic zebrafish expressing green fluorescent protein (GFP) under the LM02 promoter. Global gene expression patterns will be evaluated by microarray analysis of wild type and mutant mice and zebrafish. In situ hybridization studies performed on 6598 cDNA clones from zebrafish libraries have uncovered 128 zebrafish genes expressed in hematopoietic and vascular tissues during embryogenesis. This includes genes such as draculin, a novel zinc finger transcription factor, which is the earliest marker of ventral mesoderm fated to become blood. We plan to screen this zebrafish stem cell library by in situ hybridization to obtain new markers of stem cells and hemangioblasts. Mammalian orthologs of these zebrafish genes will be characterized. The genes will be positioned on the zebrafish genome map and correlated to known zebrafish mutations. Several factors will be studied by gene knockdown and overexpression in zebrafish, and by gene targeting experiments in murine embryonic stem cells. Transgenic mice and zebrafish will be used to evaluate gene regulatory elements predicted by a novel computer algorithm. The information developed in this application will be distributed through a WWWeb site. These studies should produce a better molecular characterization of hematopoietic and vasculogenic stem cells and could provide therapies for patients with anemia or leukemia.